Expression cloning of neurotrophic factors using Xenopus oocytes.

We have explored the potential for cloning novel neurotrophic factor cDNAs via assay of neurotrophic activities following expression in Xenopus oocytes. In this report, we describe the successful application of the method to tract rat ciliary neurotrophic factor (CNTF) activity from mRNA purified from cultured cells and from mRNA synthesized by in vitro transcription of a cDNA library. Rat C6 glioma cells, which had been previously shown to have CNTF-like activity (Westermann et al., 1988), were used as source material. We tested protein extracts of C6 cells using an in vitro assay of primary neurons from the chick ciliary ganglion (CCG assay) and detected a CNTF-like activity. RNA isolated from C6 cells was shown to direct the synthesis of the activity following microinjection into Xenopus oocytes and one-step fractionation of Xenopus extract. C6 mRNA was size-fractionated, and fractions encoding CNTF-like activity were cloned into a lambda phage vector at a site distal to a T7 promoter. Synthetic RNA transcribed from total library DNA was injected into Xenopus oocytes, and a CNTF-like activity in the oocyte extract was detected by the CCG assay. Further fractionation of library clones narrowed the presence of the clone encoding the CNTF-like activity to a pool containing 20,000 members. The presence of a full-length CNTF cDNA clone in this pool and partial clones in other pools was confirmed by Polymerase Chain Reaction (PCR) using oligonucleotides from the rabbit CNTF cDNA (Lin et al., 1989) as primers.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of CA III in rodent models of obesity.

To achieve a better understanding of the biochemical basis of obesity, we have undertaken comparative analyses of adipose tissue of lean and obese mice. By two-dimensional gel analysis, carbonic anhydrase-III (CA III) has been identified as a major constituent of murine adipose tissue. Quantitative comparisons of CA III protein and mRNA levels indicate that this enzyme is expressed at lower levels in adipose tissue from animals that were either genetically obese or had experimentally induced obesity compared to levels in the corresponding lean controls. This decrease in CA III expression was unique to adipose tissue, since other CA III-containing organs and tissues did not show a change when lean and obese animals were compared. Additionally, levels of CA III in adipose tissue from obese animals responded to acute changes in energy balance of the animal. These results are discussed in light of possible metabolic roles for CA III.

Selective reduction of mRNA for the beta-amyloid precursor protein that lacks a Kunitz-type protease inhibitor motif in cortex from Alzheimer brains.

In poly(A) RNA from cerebral cortex obtained postmortem from victims of Alzheimer's disease (AD), an alternatively spliced mRNA for the amyloid precursor protein (APP-695 mRNA) was shown to be decreased by 65%. Another form (APP-751 mRNA) with an additional exon encoding a Kunitz-type (serine) protease inhibitor motif did not change appreciably (less than 30% decrease) in AD cortex. If this twofold increase in the APP-751 mRNA/APP-695 mRNA ratio results in a corresponding increase in the APP-751/APP-695 protein ratio, this would support the hypothesis that impaired proteolysis promotes the accumulation of abnormal proteins in the brain during AD. In the two previously known, major alternatively spliced forms of ca. 3.3 and 3.5 kb, we resolved doublet RNAs for each form that are consistent with sequence data showing multiple polyadenylation sites (J. Kang et al., 1987, Nature (London) 325, 733-736.). Smaller APP-related transcripts were also found (1.1, 1.0, 0.8, and 0.3 kb), some of which are selectively altered in AD.

Biochemical similarity of expressed human prorenin and native inactive renin.

Prorenin is secreted by mammalian cells transfected with a human preprorenin expression construct. The purpose of this investigation was to compare the physicochemical properties of expressed prorenin in culture medium with the known characteristics of human inactive renin, which accounts for nearly half the renin in plasma and kidney. We found that expressed human prorenin strongly resembles human renal and plasma inactive renin. The expressed prorenin was inactive and could be equally activated by acid (dialysis to pH 3.3) or trypsin. Acid activation was completely reversible; reexposure to acid could reactivate the expressed inactive renin. Exposure to cold (-5 degrees C for 3 days) could also activate expressed renin. The Michaelis-Menten constant of acid-activated expressed renin with sheep substrate was 0.29 microM, and the pH optimum was 7.8. Expressed inactive renin bound to a cibacron-blue affinity column and could be eluted with 0.5M NaCl. All the above characteristics resemble those of human renal and plasma inactive renin. In addition, the molecular weight of expressed prorenin and human chorionic renin was 47,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 46,000, as measured by high-performance liquid chromatography. These data, taken together with the published observation that native human inactive renin cross-reacts with antibodies generated against amino acid sequences in the prosegment of renin, provide strong support for the hypothesis that human inactive renin is prorenin.

Characterization of human prorenin expressed in mammalian cells from cloned cDNA.

Human preprorenin was synthesized in Chinese hamster ovary (CHO) cells transfected with an expression vector containing renin cDNA sequences. These cells secrete an inactive form of renin (EC 3.4.23.15) that can be activated by trypsin. This inactive renin is precipitable by antibody generated against purified human renal renin and also by antisera generated to a synthetic peptide derived from the amino acid sequence of the pro segment of preprorenin (anti-propeptide), indicating that the secreted inactive enzyme is a form of prorenin. Analysis of [35S]methionine-labeled proteins immunoprecipitated from CHO cell conditioned culture medium indicates that prorenin is expressed in CHO cells as two distinct forms that differ in their degree of glycosylation. In vitro trypsin activation of prorenin cleaves approximately 4.5 kDa from the protein, rendering it unreactive with the antipropeptide antiserum but still recognizable by anti-renal renin antibody. These results show directly that the prorenin expressed by CHO cells is an inactive enzyme that is activated by trypsin cleavage of the pro segment. The ability to express human renin in this form will allow for the purification of both active and inactive forms of the enzyme in quantities sufficient for detailed physiological and structural studies.

Overproduction of immunoglobulin mRNA by a murine myeloma MOPC 315 variant cell line.

The mouse myeloma MOPC 315 cell line synthesizes and secretes IgA (lambda 2) immunoglobulin. A spontaneously arising variant of the MOPC 315 line, which had been isolated as apparently oversecreting IgA protein, has been characterized. The variant line has been shown to synthesize and secrete increased levels of heavy chain, light chain, and J chain polypeptide compared to the parental wild-type cells from which it was isolated. The steady-state levels of cytoplasmic mRNA for these polypeptides are increased commensurately in the over-producing line. For the heavy chain, enhanced transcription, and possibly increased gene dosage, appear to be involved. The increased levels of the three individual immunoglobulin polypeptide chains suggest that the variant line displays a coordinate regulation of expression of immunoglobulin genes.